Process for gibberellic acid production with “Fusarium moniliforme” strains

ABSTRACT

The present invention provides an improved process for the production of gibberellic acid (GA 3 ), achieving a yield over 225 g/kg of GA 3  with solid substrate fermentation, and over 15 g/L by submerged fermentation. The method also provides novel substrates, including the use of Jatropha seed cake. The present invention has in particular provided an improved, cost-effective process for the manufacture of GA 3 , as the process has a surprisingly high yield of product, achieves the maximal yield in shorter time than other techniques, consumes less energy, and works with very inexpensive substrates. In all, the manufacturing costs are significantly reduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Indian provisional applicationNo. 1473/MUM/2006, filed on Sep. 15, 2006, the disclosure of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an improved process for the productionof gibberellic acid by fermentation techniques. The present invention inparticular relates to the production and optimization of gibberellicacid with strains of Fusarium moniliforme by submerged or solid statefermentation.

BACKGROUND OF THE INVENTION

Gibberellic acid (GA₃) is the most important gibberellin, a class ofditerpenoid acids that function as plant growth regulators (Jefferys, E.G., Adv. Appl. Biol. 13:283-316 (1970)). GA₃ affects stem elongation,elimination of dormancy, flowering, sex expression, enzyme induction andleaf and fruit senescence. GA₃ is a high-value, industrially-important,biochemical with various applications in agriculture (L. M. Pastrana etal., “Interactions affecting gibberellic acid production in solid-stateculture: A factorial study”; Enzyme and Microbial Technology 17:784-790(1995), citing to Kumar, P. K. R., and Lonsane, B. K., Appl. Microbiol.Biotechnol. 34:145-148 (1990)). However, its high cost has restrictedits use to premium crops (Jefferys, E. G., Adv. Appl. Biol. 13:283-316(1970)).

GA₃ has been obtained by chemical synthesis, extraction from plants andby microbial fermentation. Stereospecific chemical synthesis involvesmultiple steps and the use of expensive reagents. To date, chemicalsynthesis is not economically competitive with fermentation techniques.Direct extraction of GAs from higher plants is not economically feasibledue to extremely low concentration of GAs in plant tissue. A largenumber of bacteria, actinomycetes and yeast cultures have been reportedto produce GA₃ or GA₃-like substances, but most do not produce GA₃ atcommercially-feasible levels. Today, GA₃ is produced by fermentation,mostly from fungi.

Fermentation Methods

GA₃ production in fungal culture includes liquid surface fermentation,submerged fermentation, and solid state fermentation.

Liquid Surface Fermentation (LSF)

Liquid surface fermentation (LSF), often referred to as surfacefermentation, was employed in earlier years for the production of GA₃but suffered from low yield (e.g. 3.3-9.2 g of crude powdered GA₃ from130 liters of medium) problems with scalability, contamination and more,and was discontinued (P. K. R. Kumar and B. K. Lonsane, “Microbialproduction of Gibberellins: state of the art” in Advances in AppliedMicrobiology. 34: 29-140 (1989)).

Submerged Fermentation (SmF)

Currently, GA₃ is largely produced by submerged fermentation (SmF) ofthe fungus Gibberella fujikuroi. The fungus known as Fusariummoniliforme is also used; F. moniliforme is the anamorph (asexual stage)of Gibberella fujikuroi. GA₃ is also synthesized from several bacteriasuch as Azotobacter, and Azospirillium in culture medium and from wildstrains of fungi such as Sphaceloma sp., Phaeosphaeria sp., andNeurospora sp.

The initial pH values generally employed by various workers are withinthe range 3.5-5.8, especially around pH 5.5. Id. pH was not usuallycontrolled during fermentation and thus resulted in a final pH differingfrom the starting pH, with reports of the final pH of 3.9-5.2, 1.8-1.9,or even slight alkalinization. Id.

The effect of temperature on the production of GA₃ is dependent on thestrain employed. The optimal temperatures reported for the production ofGA₃ using G. fujikuroi or F. moniliforme include 25° C.; 28.5-29.5° C.;30° C.; 34° C.; 29^(±) 0.5° C.; 27° C.; and 28° C. (See Id.)

Different workers have used a variety of carbon sources for theproduction of GAs, as reported by different authors. A combination ofreadily and slowly metabolizable carbon sources gave a higher yield ofGA₃. Id. Use of molasses led to decreased but economically usefulyields. Id. There was a 49% decrease in the yield of GAs with stearicacid as compared to sucrose. Use of dairy waste as carbon sourceresulted in 0.75 g GA₃/liter in 12 days. The production was completelyinhibited by 1 ppm geraniol due to total inhibition of cell growth. Adirect proportional relationship has been shown between the initialconcentration of nitrogen supplied in the medium and the rate of productformation as well as the amount of metabolite produced. Id. See alsoJefferys, E. G., Adv. Appl. Biol. 13:283-316 (1970)

Trace elements such as Fe, Cu, Mn, Zn, Al and Ca are required in thefermentative production of GAs. Id. Trace elements may be expresslyadded in excess or in combination, as in Raulin-Thom medium, orsufficient trace elements may be present as impurities in otheringredients of the fermentation medium. Id.

Other growth factors such as vitamins or Yeast extract may improveyield; while other heavy metals or the use of lines steel or stainlesssteel fermentation tanks can decrease yield. Id.

Various processes for production of GA₃ are described in U.S. Pat. Nos.2,842,051, 2,865,812, 2,906,671, 2,906,673 and 3,021,261. U.S. Pat. No.2,865,812 reported a yield of 630 mg/L in 664 hrs. Eleazar et al.(“Optimization of gibberellic acid production by immobilized Gibberellafujikuroi mycelium in fluidized bioreactors”, Journal of Biotechnology,76, 147-155 (2000)) reported yields by submerged fermentation as high as2.862 g/L.

Solid Substrate Fermentation (SSF)

SSF (Solid Substrate Fermentation) is defined as any fermentationprocess performed on a non-soluble material that acts both as physicalsupport and source of nutrients, in the absence of free flowing liquid.No free-moving water is present, but there is enough moisture presentfor the growth and metabolism of the microorganism. The low moisturecontent means that fermentation can only be carried out by a limitednumber of microorganisms, mainly yeasts and fungi, although somebacteria have been used. Work on the production of GAs using SSFtechnique was initiated in the early 1980s and the initial studies gavevariable yields. No information was available on the economics of theprocess until early 1987. Three cases were examined wherein the yield ofGA₃ under the SSF technique were 0.825, 1.05 and 1.54 g/kg of Dry MouldyBran. The yield of GA₃ in the fed-batch SSF Process was 1.54 g/kg of DryMouldy Bran as compared to 1.05 g/kg of Dry Mouldy Bran in the batch SSFprocess. Id. One group reported a yield of 3 g/kg by SSF (Bandelier, S.,Renaud, R., and Durand, A., “Production of Gibberellic acid by Fed-batchsolid state fermentation in an aseptic pilot-scale reactor”, ProcessBiochemistry, 32:141-145 (1997)). B. Tudzynski (“Biosynthesis ofgibberellins in Gibberella fujikuroi: biomolecular aspects”; Appl.Microbiol Biotechnol (1999) 52:298-310) reported that German PatentNumber DD252000 also described processes for GA₃ by submergedfermentation, and reported yields of 8 g/kg.

Art-Known Fermentation Techniques Remain Expensive

Despite advances in fermentation technology, the cost of production ofGA₃ has been a deterrent to its widespread use. Additional costconsiderations include problems in the downstream processing and, giventhe potency of GA₃ to plants, removal and disposal of contaminatedwastewater.

SUMMARY OF THE INVENTION

The inventors of the present invention have developed a process tomanufacture GA₃ with Fusarium moniliforme by submerged and solid statefermentation and have been successful in obtaining yields over 15 g/L insubmerged fermentation, and over 200 g/kg in solid state fermentation.

In one embodiment of the present invention, GA₃ is produced by solidstate fermentation using various substrates such as wheat bran andJatropha seed cake, to which mineral salts are added under high moisturecontent. The mixture is further inoculated with F. moniliforme andincubated for 10 days and the content of the mixture is analyzed forGA₃. GA₃ is isolated by adjusting the aqueous dilution of the mixture toacidic pH, and extracting using an organic solvent. The organic solventis distilled and the GA₃ obtained is dissolved in ethanol.

In another embodiment of the present invention, GA₃ is produced bysubmerged fermentation by culturing F. moniliforme in Czapek-Dox mediacontaining a carbon source, and incubating for 10 days.

The present invention has provided industrially-viable processes for themanufacture of GA₃ by fermentation processes such as solid-statefermentation or submerged fermentation. The present invention has inparticular provided an improved, cost-effective process for themanufacture of GA₃, as the process has a surprisingly high yield ofproduct, achieves the maximal yield in shorter time than othertechniques, consumes less energy, and works with very inexpensivesubstrates. In all the manufacturing costs are significantly reduced.

In one embodiment, the present invention has focused on production andoptimization of gibberellic acid by Fusarium moniliforme using variousfermentation techniques.

The present disclosure provides GA₃ produced either by submerged orsolid-state fermentation (SSF) technique. In certain embodiments, thesubmerged fermentation is carried out in reconstituted Czapek-Dox brothwhereas solid-state fermentation is done on a humid solid matrix.

In another embodiment, the present invention relates to the use ofmicroorganism belonging to the genus Gibberella, including Gibberellafujikuroi or Fusarium moniliforme.

Production of GA₃ is influenced by cultural conditions. In certainembodiments, high yields of GA₃ are obtained by varying factors, such aspH, temperature, incubation time, and other conditions such asoptimization of the fermentation media.

In another embodiment, the present invention may use humid solidmatrices substrates such as wheat bran or Jatropha seed cake.

In another embodiment, the production process of GA₃ by submergedfermentation involves incubating the fungi in liquid media containingvarious minerals, sodium nitrate as the nitrogen source, and sucrose asthe carbon source.

In another embodiment, the present invention has optimized the pHconditions for submerged fermentation. The process of the presentinvention provides an optimal pH in the range of 5-8. In one embodiment,the pH is pH 7.0

In another embodiment, the present invention has optimized thetemperature conditions for submerged fermentation. The present inventionprovides an optimal temperature ranging between 25° C. (roomtemperature) to 37° C. In one embodiment, the temperature is 30° C.

In another embodiment, the present invention has optimized the carbonsource for submerged fermentation. In one embodiment, the carbon sourceis sucrose or glucose.

In another embodiment, the present invention has optimized the media forsubmerged fermentation. In one embodiment, the medium is reconstitutedCzapek-Dox medium.

In still another embodiment, the present invention provides a processfor production of GA₃ by solid-state fermentation. In one embodiment,this involves growing fungi on wheat bran supplemented with a mineralsalts solution.

In the case of GA₃ production in SSF, factors that affect yield includetemperature, pH, moisture, substrate particle size, light, autoclavetiming, incubation period, amount and age of inoculum.

GA₃ was produced by submerged or solid-state fermentation (by free orimmobilized cells, respectively). We observed that the yield fromsolid-state fermentation was as high as 39 g/kg using wheat bran as asubstrate, and 237.2 g/kg, when using of Jatropha seed cake assubstrate. To the best knowledge of the inventors, the GA₃ yields of thepresent invention are higher than any reported, by any mode offermentation. For example, only one previous report provides a yield ashigh as 19.3 g/kg of GA₃, which was obtained using starch (corn flour)as substrate (Gelmi et al. (“Solid substrate cultivation of Gibberellafujikuroi on an inert support”; Process Biochemistry. 35: 1227-1233(2000), citing to Qian et al., (World J. Microbiol Biotechnol. 10:93-98,(1994))).

A comparison of the present invention with conventional methods wasperformed. GA₃ produced by the present invention by submerged is greaterthan 3-fold higher than the highest yield reported in the literature;and by solid-state fermentation greater than 10-fold higher. The presentprocess has demonstrated a yield greater than 15 g/L by submergedfermentation, and greater than 225 g/kg by solid state fermentation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.

FIG. 1: Production of GA₃ production (g/kg) by F. moniliforme in SSFusing Jatropha seed cake as the substrate.

FIG. 2: Illustrates GA₃ production in g/L and biomass, as determined byOD at 600 nm, for Fusarium moniliforme grown by submerged fermentationin commercial Czapek-Dox broth.

FIG. 3: Illustrates the effect of pH on GA₃ production (g/L) and biomass(OD at 600 nm) by F. moniliforme in submerged fermentation in commercialCzapek-Dox broth.

FIG. 4: Illustrates the effect of temperature on GA₃ production by F.moniliforme in submerged fermentation in Czapek-Dox broth.

FIG. 5: Illustrates the effect of carbon source on GA₃ production by F.moniliforme in submerged fermentation in Czapek-Dox broth. No growth wasobserved when acetic acid was used as the carbon source.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the term GA₃ or gibberellic acid refers to gibberellicacid with the molecular weight of 346.38 g/mol.

As used herein the term “solid state fermentation (SSF)” includes aprocess wherein the microbial growth and formation of product on andinside a humid solid matrix is in the absence of free water. This isalso known as “solid substrate fermentation.

As used herein the term “submerged fermentation” includes a processwherein microbe is grown in a liquid medium and the product is secretedinto the media.

The mineral salts solution used in the present invention comprisescopper sulphate, ferric chloride and zinc sulphate.

Czapek-Dox (Himedia-M076) media contains the following components (perliter)

sucrose 30 g sodium nitrate 3 g dipotassium phosphate 1 g magnesiumsulphate 0.5 g potassium chloride 0.5 g ferrous sulphate 0.01 gdistilled water 1000 ml

In one embodiment, the Czapek-Dox media is reconstituted rather thancommercially purchased. Reconstituted Czapek-Dox media is prepared byadding all components individually in water at the same concentrationsas that of commercially available media.

One substrate used in the present invention is Jatropha seed cake.Jatropha is grown for its oil, which is extracted after crushing theseeds. Typically, seeds are ground to uniform sized particles and oil isextracted by Soxhlet extraction using Hexane as a solvent. Jatropha seedcake “with oil” means that the seeds have been crushed, but before theoil has been extracted. Seed cake “without oil” refers to seed cake leftover after oil extraction, from which more than about 95% of the oil hasbeen removed.

Solid State Fermentation

The present disclosure provides an improved process for the productionof GA₃ either by submerged fermentation or solid state fermentation.

In one embodiment, GA₃ production via solid state fermentation comprisesthe following steps:

a) Preparation of solid substrate mixture;

b) Inoculation with F. moniliforme culture;

c) Incubation;

d) Extraction of GA₃; and

e) Purification.

In certain embodiments, the solid-state substrate used in the presentinvention is wheat bran (50 g) or Jatropha seed cake (5 g) to which isadded mineral salt solutions (30 ml and 8 ml, respectively) under highmoisture content and then autoclaved in the flasks. In one embodiment,autoclaved substrate is inoculated with F. moniliforme culture inCzapek-Dox broth.

In one embodiment, incubation of the substrate is done at temperaturesranging between 25-37° C. and analyzed for GA₃ content, at periodicintervals, using a spectrophotometric method (Berrios et al.(Spectrophotometric method for determining Gibberellic acid infermentation broths. Biotechnology Letters, 26: 67-70 (2004)),high-pressure liquid chromatography (HPLC) and/or thin-layerchromatography (TLC). In TLC the culture filtrate obtained afterfermentation was subjected to extraction and purification as describedabove. The residue obtained was dissolved in ethanol and separated bythin layer chromatography using isopropanol-ammonia-water (10:1:1,v/v/v) as mobile phase. The plates were sprayed with 3% sulphuric acidin methanol containing 50 mg ferric chloride and heated in an oven at80° C. for 10 min. GAs fluoresce and appear as greenish spot under UVlight, allowing their detection. (D. Puchooa and R. Ramburn; “A study onthe use of carrot juice in the tissue culture of Daucus carota: AfricanJournal of Biotechnology, Vol. 3(4), pp. 248-252, April-2004).

The inventors of the present invention extracted GA₃ by diluting theabove mixture with water, and acidification with concentrated HCl, to pH2.5. To 5 ml of broth, 60 ml of methanol:chloroform:2 N ammoniumhydroxide in the ratio of 12:5:3 and 25 ml of distilled water was added.The mixture was shaken well in a separating funnel. The bottomchloroform layer was removed and methanol in the upper aqueous layer wasevaporated. After adjusting the pH of the remaining solution to 2.5, thesolution was extracted thrice with 15 ml of ethyl acetate each time. Theethyl acetate phase was collected and evaporated to dryness. The driedmaterial was dissolved in 5 ml of ethanol and the amount of GA₃ wasdetermined. To a 1 ml aliquot of the sample in ethanol, 8 ml of 3.75NHCl was added and O.D. was measured at 254 nm after 2 min., followingthe protocol of Berrios et al. (2004).

Various substrates have been used for solid state fermentation in theliterature. Machado et al. reported a yield of 0.925 g of GA₃/kg ofbiomass using coffee husk or cassava bagasse as a medium (Machado, C. M.M., Oishi, B. O., Pandey, A., and Soccol, C. R., Biotechnol. Prog.20:449-1453 (2004)). Gelmi et al. (“Solid substrate cultivation ofGibberella fujikuroi on an inert support”; Process Biochemistry. 35:1227-1233 (2000)) reported that Qian et al., (World J. Microbiol.Biotechnol. 10:93-98, (1994)) achieved a yield of 19.3 g GA₃/kg of dryfermented substrate after 18 days of cultivation using corn flour as asubstrate (although Gelmi suggested that the yield was actually less,when taking into account degradation of the substrate).

Gelmi, ibid. also reported that other workers achieved yields of 3.8 gGA₃/kg vermiculite and of 6.8 g GA₃/kg initial dry mass over 190 h,using a wheat bran culture medium. A yield of 8 g/kg of GA₃ was obtainedon rice as a substrate (German Patent Number DD 252000). Prema et al.,Indian J. Microbiol. 28:1-2 (1988)) reported very low yields of GA₃(1.14 g/kg) and Kumar, P. K. R., and Lonsane, B. K., Appl. Microbiol.Biotechnol. 34:145-148 (1990), reported 1.2 g/kg of GA₃ using wheat branas a medium.

The present invention has obtained GA₃ using wheat bran, and usingJatropha seed cake with oil and without oil. The present invention hasfound that the GA₃ yields were as high as 237.2 g/kg using Jatropha seedcake with oil on the 6^(th) day, which is more than ten fold higher thanthe previously reported possible yield of 19.3 g/kg (Gelmi, ibid.). TheGA₃ yield obtained by using Jatropha seed cake as a substrate was alsofive fold higher than that obtained by wheat bran under the presentoptimized conditions (see Examples).

In certain embodiments, the yield of GA₃ in the present invention isfurther improved by having a higher initial moisture level of 60% and alower incubation temperature of 23° C. as compared to moisture level of50% at 28° C. or 30° C., as reported in Prema et al., Indian J.Microbiol. 28:1-2 (1988), or Pastrana et al., Enzyme and MicrobialTechnology, 17:784-790, 1995 (1995). There does exist one report whereinthe temperature was decreased from 28° C. to 22° C. as the fermentationprogressed. However, the yield of GA₃ was only 3 g/kg (Bandelier et al.,Process Bioche. Vol. 32 2:141-145 (1997)).

Submerged Fermentation

In one embodiment, the present invention provides a process forsubmerged fermentation which comprises the following steps:

1. Preparation of the inoculum;

2. Incubation in medium containing sugars;

3. Extraction of GA₃; and

4. Purification.

In one embodiment, the inoculum is prepared by culturing and maintainingF. moniliforme in Czapek-Dox broth medium, in the presence of a carbonsource, such as sucrose or glucose.

In the present invention, various conditions such as pH, temperature,carbon source, and the media may be optimized for high yield.

One group has previously reported a yield of GA₃ of 5 g/L by submergedfermentation, using a fed-batch cultivation mode under conditions ofnitrogen limitation using genetically improved strains (P. K. R. Kumarand B. K. Lonsane, “Microbial production of Gibberellins: state of theart” in Advances in Applied Microbiology. 34: 29-140 (1989)).

General Comments

The following examples are included to demonstrate certain exemplaryembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constituteexemplary modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLES Example 1 Organism

Fusarium moniliforme NCIM 1100 was obtained from National Collection ofIndustrial Microorganisms, Pune, India. The strain was cultured andmaintained on sterile potato dextrose agar (PDA) slants. Unlessotherwise indicated, the media used for production of gibberellic acidwas Czapek-Dox (Himedia-M076) and the culture was incubated at 30° C.for 10 days on as shaker incubator at 150 rpm.

Example 2 Solid Substrate Fermentation with Wheat Bran

To 50 g wheat bran, 30 ml of a mineral salts solution (copper sulphate,0.007 g; ferric chloride, 0.007 g; and zinc sulphate, 0.007 g, dissolvedin 1 liter of 0.2N HCl) was added and mixed well. The mixture was thendistributed equally into five flasks and sterilized at 15 PSI for 30min. The autoclaved wheat bran in each flask was inoculated with 10 mlof 6 days old F. moniliforme culture from Czapek-Dox broth, mixedthoroughly and were incubated at 30° C. for 10 days at a 45° angle.Yield was determined by the method of Berrios et al., as describedbelow.

Yield of GA₃ from wheat bran under SSF, over time. GA₃ concentration(g/kg) Days of in solid substrate incubation fermentation (wheat bran) 529 6 32 7 33 8 34 9 39 10 39.4

Example 3 Analytical Procedures

GA₃ was determined spectrophotometrically by the method described byBerrios et al. (Biotechnology Letters 26: 67-70, (2004)) at 254 nm. GA₃was also determined by HPLC at 206 nm using a C18 column withmethanol:water (3:1) as the mobile phase at 1 ml/min flow rate, asdescribed by Sharma et al. (Biotechnol. Appl. Biochem., 39, 83-88(2004)). The retention time of GA₃ is 3 minutes under these conditions.

GA₃ was also detected by TLC as described by Puchooa et al. (AfricanJournal of Biotechnology, 3(4): 248-252 (2004)). The culture filtrateobtained after fermentation was subjected to extraction and purificationas described below. The obtained residue was dissolved in ethanol andseparated by TLC using isopropanol-ammonia-water (10:1:1, v/v/v) asmobile phase. The plates were sprayed with 3% sulphuric acid in methanolcontaining 50 mg ferric chloride and heated in an oven at 80° C. for 10min. GA₃ fluoresce and appear as a greenish spot under UV light.

Example 4 Extraction of GA₃ from the Wheat Bran Solid Substrate

GA₃ was extracted from the solid substrate by adding 100 ml of distilledwater to moldy bran in each flask and were kept on shaking incubator at150 rpm for 2 hrs. The slurry from each flask was filtered throughmuslin cloth and the volume of the filtrate was made to 100 ml. Filtratewas centrifuged at 10000 rpm for 10 min at 28° C. Supernatant wascollected and analyzed for GA₃ concentration spectrophotometrically.

Example 5 Purification of GA₃

Isolation of GA₃ from the solid substrate extract was done by the methoddescribed in Ergun et al. (Turk J. Bot., 26: 13-18 (2002)). Briefly, to5 ml of the water extract from the solid substrate, 60 ml ofmethanol:chloroform:2 N ammonium hydroxide in the ratio of 12:5:3 and 25ml of distilled water was added. The mixture was shaken well in aseparating funnel. The bottom, chloroform, layer was removed andmethanol in the upper aqueous layer was evaporated. After adjusting thepH of the remaining solution to 2.5 with concentrated HCl the solutionwas extracted thrice with 15 ml of ethyl acetate. The ethyl acetatephase was collected and evaporated to dryness. The dried material wasdissolved in 5 ml of ethanol and GA₃ was determined, using the protocolof Berrios et al. This method of purification may also be adapted to GA₃from submerged fermentation.

Example 6 Solid Substrate Fermentation with Jatropha Seed Cake

Jatropha seed cake, one of cheapest substrates available, was employedas the substrate for the production of GA₃ Production of gibberellicacid using Jatropha as the substrate has never been previously reported.Both Jatropha seed cake with oil and without oil were used as thesubstrate. Five flasks (5 g of Jatropha, 8 ml of MSS (copper sulphate0.007 g; ferric chloride 0.007 g; zinc sulphate: 0.007 g; dissolved in 1liter of 0.2N HCl) and 3.5 ml of inoculum)) were inoculated with 3.5 mlof four day old inoculum of F. moniliforme, and were incubated at roomtemperature. Growth was observed within 24 hours of inoculation. Maximumyield was obtained on the 6^(th) day of incubation and the yield wasfound to decrease on the 8^(th) and 10^(th) day.

As depicted in FIG. 1, the maximum yield of GA₃ obtained was 237.2 g/kgby SSF. This is so far the best-reported yield of GA₃ obtained by SSF.The GA₃ extracted from the SSF migrated on TLC as a single spot similarto the standard and showed an R_(f) value of 0.74, consistent with GA₃.

Example 7 Production of GA₃ in Jatropha Seed Cake without Oil

The production of gibberellic acid was determined every 48 hours. Fiveflasks (5 g of Jatropha seed cake without oil, 8 ml of mineral saltssolution MSS and 3.5 ml of inoculum) were inoculated with 3.5 ml of fourday old inoculum (F. moniliforme) and was incubated at room temperature.Maximum yield was obtained on the 6^(th) day of incubation and the yieldwas found to decrease on the 8^(th) and 10^(th) day.

Days of GA₃ Concentration incubation g/kg 2 122 4 136 6 213 8 145 10 159

Example 8 Comparison of Yield from Jatropha Seed Cake with Oil andwithout Oil

Gibberellic acid was produced by using Jatropha seed cake with oil andwithout oil as the substrates. A very slight difference was observed inthe yield, however yield from Jatropha seed cake with oil (149.72 g/kg)was found to be slightly higher than the yield obtained from Jatrophaseed cake without oil (140.22 g/kg) on day 10 after inoculation.

Example 9 Submerged Fermentation

Fusarium moniliforme culture was inoculated from PDA slants into 250 mlof commercial Czapek-Dox broth at an initial pH of 7.0, and incubated at30° C. for 10 days on a shaking incubator at 150 rpm. Cell growth wasmonitored by collecting 1 ml of culture from fermented broth every 24 h,and centrifuged at 13200 rpm for 10 min. The supernatant was used forGA₃ estimation, and the pellet, washed thrice with saline, was used fordetermining cell growth. The growth and GA₃ production patterns of F.moniliforme culture in Czapek-Dox broth is shown in FIG. 2.

After an initial lag, there was an exponential increase in growth. Thislog phase continued up to 5 days before reaching a plateau indicatingthe commencement of stationary phase. Production of GA was found tostart increasing during the late exponential phase (5 days) andextending well into the stationary phase before reaching a plateau (FIG.2). Under these conditions, the yield of GA₃ reached the highest on the8^(th) day.

Days of F. moniliforme inoculum Incubation GA₃(g/L) Biomass 1 0.09 1.502 — — 3 1.92 14.70 4 2.76 19.44 5 3.29 23.84 6 3.35 24.36 7 3.71 27.20 84.21 22.24 9 — — 10 4.02 24.56

Example 10 Optimization of pH in Submerged Fermentation

Optimization studies were carried out to increase the yield of GA₃. Todetermine the optimal pH for the growth and production of gibberellicacid, Fusarium moniliforme culture was inoculated into 100 ml of brothin 250 ml flasks, with flasks having different initial pH values (5, 7and 8). The cultures were incubated at 30° C. for 10 days on a shakingincubator at 150 rpm. Sampling of 1 ml of broth was done every 24 hthroughout the incubation period, to determine cell growth and GA₃concentration. Initial pH did not have any significant effect on the GA₃yield (FIG. 3) although the highest yield (6.5 g/L) was obtained at aninitial pH of 7.0.

Days of pH 5 pH 7 pH 8 Incubation GA₃(g/L) GA₃(g/L) GA₃(g/L) 1 0.10 0.140.11 2 — — — 3 1.15 1.32 1.24 4 2.43 2.99 2.57 5 3.59 4.45 3.48 6 4.254.86 3.79 7 4.50 5.31 4.22 8 5.10 6.50 4.73 9 — — — 10 5.46 5.87 5.02

Example 11 Optimization of Temperature in Submerged Fermentation

The optimal temperature for the growth and production of gibberellicacid by Fusarium moniliforme was evaluated. The F. moniliforme culturewas inoculated into four different Czapek-Dox broth flasks, which wereincubated at different temperatures (25° C., 30° C., 37° C., and roomtemperature of 23-25° C.) for 10 days on a shaking incubator at 150 rpm.Sampling was done after every 24 hrs for determination of cell growthand GA₃ concentration. Incubation at 30° C. was the optimal conditionfor maximizing the production of GA₃ (FIG. 4).

Days of 25° C. 30° C. 37° C. 23° C. Incubation GA₃(g/L) GA₃(g/L)GA₃(g/L) GA₃(g/L) 1 0.10 0.11 0.024 0.10 2 — — — — 3 1.14 1.32 0.0921.21 4 2.33 2.97 0.10 2.73 5 3.0 4.06 0.29 3.33 6 3.10 4.34 0.33 3.86 73.34 4.76 0.32 4.57 8 3.88 5.77 0.23 5.01 9 — — — — 10 3.65 5.42 0.21.92

Example 12 Optimization of Carbon Source in Submerged Fermentation

To determine the carbon source, which gives optimal yield of GA₃production, the following carbon sources were used: sucrose, glucose,galactose, xylose, glacial acetic acid and methanol. Fusarium culturewas inoculated into reconstituted Czapek-Dox medium containing theaforementioned carbon sources and incubated at 30° C. for 10 days on ashaking incubator at 150 rpm. Sampling was done every 24 hrs throughoutthe incubation period for determining the cell growth and the GA₃concentration. Sucrose and glucose were found to be the best source ofcarbon giving a yield of over 15 g/L (FIG. 5). Galactose and xylose alsogave yields higher than 8 g/L.

GA₃ (g/L) Glacial Days of Acetic Incubation Glucose Galactose XyloseMethanol acid Sucrose 1 0.28 0.23 0.64 0.026 No 0.43 Growth 2 1.12 0.890.66 0.028 No 1.72 Growth 3 3.07 2.57 1.61 0.018 No 5.57 Growth 4 5.634.89 3.51 0.015 No 7.69 Growth 6 12.14 10.93 7.81 0.014 No 13.85 Growth7 14.02 12.17 9.25 0.001 No 14.6 Growth 8 13.82 12.35 11.08 0.0 No 14.76Growth 9 15.07 12.64 11.58 0.023 No 15.37 Growth 10 15.28 13.3 12.450.018 No 15.91 Growth

Example 13 Optimization of Submerged Fermentation

A Fusarium moniliforme culture was inoculated into reconstitutedCzapek-Dox medium, followed by incubation at 30° C. at an initial pH of7.0, using sucrose as the carbon source, and was incubated for 10 dayson a shaking incubator maintained at 150 rpm. Sampling was done every 24h throughout the incubation period for determining the cell growth andthe GA₃ concentration. As shown in the following table, the yieldreached 15.9 g/L at 10 days.

Days of incubation GA₃(g/L) 1 0.43 2 1.72 3 5.57 4 7.70 5 — 6 13.85 714.60 8 14.76 9 15.37 10 15.91Discussion: Yield from Submerged Fermentation and Solid SubstrateFermentation.

The GA₃ yield was determined in both submerged fermentation and solidsubstrate fermentation. The yield obtained from solid substratefermentation reached as high as 237.2 g/kg when using Jatropha seed cakeas a substrate, and as high as 39.4 g/kg when using wheat bran as asubstrate. The yield obtained from submerged fermentation reached ashigh as 15.9 g/L.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are chemically or physiologicallyrelated may be substituted for the agents described herein while thesame or similar results would be achieved. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. A process for producing gibberellic acid (GA₃) comprising: (a)growing Fusarium moniliforme by solid-state fermentation in the presenceof Jatropha seed cake as a substrate; and (b) obtaining gibberellicacid; wherein the yield of gibberellic acid is at least 30 g gibberellicacid/kg of substrate.
 2. The process of claim 1, wherein the yield ofgibberellic acid is at least 100 g gibberellic acid/kg of substrate. 3.The process of claim 1, wherein the yield of gibberellic acid is atleast 200 g gibberellic acid/kg of substrate.
 4. The process of claim 1,wherein the Jatropha seed cake substrate is with oil.
 5. The process ofclaim 1, wherein the Jatropha seed cake substrate is without oil.
 6. Theprocess of claim 1, wherein the solid-state fermentation is carried outin the presence of a mineral salt solution.
 7. The process of claim 1,wherein the solid-state fermentation is stopped after 6 days.
 8. Theprocess of claim 1, wherein initial moisture level is 60%, and thetemperature is 23° C. in step (a).
 9. The process of claim 1, whereinthe process further comprises inoculating Fusarium moniliforme in thepresence of the Jatropha seed cake substrate and a mineral salt solutioncomprising copper sulfate, ferric chloride and zinc sulphate.